This invention is generally in the area of methodologies for measuring binding reactions, specifically between drugs and drug receptors using fluorescent labels.
Radioligand binding techniques have been widely used for more than a decade to study receptor pharmacology and physiology. These methodologies provide a rapid, efficient means for drug discovery and identification of putative endogenous substances that may physiologically subserve receptors, as reviewed by Yamamura, H. I., Enna, S. J., and Kuhar, M. J., eds In Neurotransmitter Receptor Binding, 2nd ed. (Raven Press, New York 1988) and Snyder, S. H. "Drug and neurotransmitter receptors. New perspectives with clinical relevance." J. Am. Med. Assoc. 261, 3126-3129 (1989). These studies are particularly useful in characterizing drug-receptor interactions and specificities.
For example, in J. Chem. Neuroanatomy, 3:59-76 (January 1990), Olsen, R. W., R. T. McCabe, and J. K. Wamsley, described the characterization of GABA.sub.A receptor subtypes using autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system. The regional distribution of radioactive ligand binding in rat brain for the different receptors of the gamma-aminobutyric acid.sub.A (GABA.sub.A)-benzodiazepine receptor/chloride channel complex was measured on tissue sections by autoradiography. Seven ligands were employed including [.sup.3 H]muscimol for high-affinity GABA agonist sites; [.sup.3 H]bicuculline methochloride and [.sup.3 H]SR-95531 for the low-affinity GABA sites; [.sup.3 H]flunitrazepam for benzodiazepine sites, and [.sup.3 H]2-oxo-quazepam for the `BZ1`-type subpopulation; and [.sup.35 S]t-butyl bicyclophosphorothionate (TBPS) and [.sup.3 H]t-butyl bicyclo-orthobenzoate (TBOB) for convulsant sites associated with the chloride channel. Allosteric interactions of benzodiazepine receptor ligands with [.sup.35 S]TBPS binding were also examined in membrane homogenates. Comparison of 19 brain regions indicated areas of overlap between these ligands, but also significant lack of correspondence in some regions between any two ligands compared. Significant differences were observed in comparing GABA agonists with antagonists, one antagonist with another, GABA ligands with benzodiazepine or convulsant sites, and even between the two convulsants TBPS and TBOB. It is likely that the characterization of receptors as subtypes based on binding alone indicate a pharmacological heterogeneity that might be exploited with subtype-specific drugs showing desirable clinical profiles.
As described by R. T. McCabe, D. R. Mahan, R. B. Smith, and J. K. Wamsley in Pharmacology Biochem. Behavior 37: 365-370 (May 1990), the binding of the triazolobenzodiazepine [.sup.3 H]alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. Studies using nonequilibrium and equilibrium binding conditions for [.sup.3 H]alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of [.sup.3 H]alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. GABA enhanced [.sup.3 H]alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bound to other receptor sites had a very weak or negligible effect on [.sup.3 H]alprazolam binding.
Despite the usefulness and sensitivity of radioligand binding techniques, the use of alternative methods to study ligand-receptor interactions may provide information not readily accessible by conventional radioreceptor techniques and circumvent some of the drawbacks (such as high cost, disposal, and potential health hazard) associated with this methodology.
Fluorescence techniques have successfully been employed to study the behavior of ligand-protein interactions. For example, fluorescent labeled substrates and antigens have proven valuable in the examination of substrate-enzyme and antigen-antibody interactions.
Several attempts have been made to use fluorescent compounds to characterize receptors. Ligands with fluorescent moieties were prepared for .alpha.-adrenergic (Correa, F. M. A., et al., Neurosci. Lett. 16, 47-53 (1980)); .beta.-adrenergic (Atlas, D., and Levitzki, A. Proc. Natl. Acad. Sci. U.S.A. 74, 5290-5294 (1977); Henis, Y. I., et al., Proc. Natl. Acad. Sci. U.S.A. 79, 2907-2911 (1982); and Rademaker, B., et al., Res. Commun. Chem. Pathol. Pharmacol. 60, 147-159 (1988)); opioid (Correa, et al., (1980) and Kolb, V. M., et al., Life Sci. 33, 423-426 (1983)); adenosine (Jacobson, K. A., et al., Biochem. Pharmacol. 36, 1697-1707 (1987)); glucagon (Heithier, H., et al., Biochim. Biophys. Acta 971, 298-306 (1988) and Ward, L. D., et al., Biochim. Biophys. Acta 971, 307-316 (1988)); steroid (Carlson, K. E., et al., J. Steroid Biochem. 32, 345-355 (1988)); and dopamine (Monsma, F. J., Jr., et al., J. Neurochem. 52, 1641-1644 (1989)) receptors.
Although many of the ligands with fluorescent moieties were reported to have moderate to high affinities when evaluated with radioligand assays, quantitation and visualization of ligand-receptor interactions by direct fluorescence measurement have been problematic. For example, investigations using fluorescent ligands to identify receptors were equivocal owing to high levels of tissue autofluorescence and apparent lack of specificity (Correa, F. M. A., et al., (1980), Rademaker, B., et al., (1988), (Hess, A. Brain Res. 160, 533-538 (1979); Barnes, P., et al., Brain Res. 181, 209-213 (1980); Rademaker, B., et al., Eur. J. Pharmacol. 111, 31-36 (1985); and Rademaker, B., et al., J. Recept. Res. 5, 121-131 (1985)). Carlson, et al. (1988) described an ethyl acetate extraction technique to analyze fluorescent ligands for steroid receptors. Nevertheless, direct quantitation of ligand-receptor interactions with fluorescent ligands has not been demonstrated conclusively.
It is therefore an object of the present invention to provide a method and reagents for use in directly quantitating ligand-receptor interactions with fluorescent ligands.
It is a further object of the present invention to provide a method and reagents for use in determining ligand-receptor interactions intracellularly and extracellularly.